scholarly journals GENERATION OF CYTOTOXIC LYMPHOCYTES IN MIXED LYMPHOCYTE REACTIONS

1973 ◽  
Vol 138 (3) ◽  
pp. 672-685 ◽  
Author(s):  
James Forman ◽  
Göran Möller
Keyword(s):  
Effector Cell ◽  
Close Contact ◽  
Test System ◽  
Adherent Cell ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Effector Cells ◽  
Cytotoxic Effector ◽  
Mixed Lymphocyte Reactions

Generation of cytotoxic effector cells by a unidirectional mixed lymphocyte reaction (MLR) in the mouse H-2 system was studied using labeled YAC (H-2a) leukemia cells as targets. The responding effector cell displayed a specific cytotoxic effect against target cells of the same H-2 genotype as the stimulating cell population. Killing of syngeneic H-2 cells was not observed, even when the labeled target cells were "innocent bystanders" in cultures where specific target cells were reintroduced. Similar results were found with spleen cells taken from mice sensitized in vivo 7 days earlier. The effector cell was not an adherent cell and was not activated by supernatants from MLR. The supernatants were not cytotoxic by themselves. When concanavalin A or phytohemagglutinin was added to the cytotoxic test system, target and effector cells were agglutinated. Under these conditions, killing of H-2a target cells was observed in mixed cultures where H-2a lymphocytes were also the effector cells. These findings indicate that specifically activated, probably thymus-derived lymphocytes, can kill nonspecifically once they have been activated and providing there is close contact between effector and target cells. Thus, specificity of T cell killing appears to be restricted to recognition and subsequent binding to the targets, the actual effector phase being nonspecific.

scholarly journals In Vitro and In Vivo Characterization of CD19/CD3 Tandab AFM11 and CD19/CD16A Tandab AFM12 Targeting NHL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2763-2763
Author(s):  
Xing Zhao ◽  
Narendiran Rajasekaran ◽  
Uwe Reusch ◽  
Michael Weichel ◽  
Kristina Ellwanger ◽  
...  
Keyword(s):  
T Cell ◽  
Target Cell ◽  
Binding Sites ◽  
Effector Cell ◽  
Nk Cell ◽  
Target Cells ◽  
Tumor Target ◽  
Effector Cells

Abstract Introduction: CD19 is expressed by B cells from early development through differentiation into plasma cells, and represents a validated target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Different CD19-targeting T-cell engagers are investigated in clinical studies for the treatment of NHL or ALL, including Affimed's AFM11, a bispecific CD19/CD3 TandAb antibody, which is currently investigated in a phase 1 dose escalation study. Indeed, Affimed's bispecific tetravalent platform comprises not only T-cell engaging TandAbs with two binding sites for CD3, but also NK-cell recruiting TandAbs with two binding sites for CD16A. In the present study, Affimed's AFM11, was characterized and compared in in vitro and in vivo studies with the CD19/CD16A TandAb AFM12. Methods: Analogous to the CD19/CD3 TandAb AFM11, a bispecific tetravalent TandAb AFM12 was constructed with two binding sites for CD19 and two sites for CD16A. Both TandAbs were characterized side by side for their biophysical properties, binding affinities to CD19+ tumor target cells and to their respective effector cells by flow cytometry. Kinetics and dose-response characteristics were evaluated in in vitro cytotoxicity assays. Potency and efficacy of both TandAbs were compared on different CD19+ tumor target cell lines using primary human effector cells. To compare the efficacy of AFM11 and AFM12 a patient-derived tumor xenograft model was developed. Results: AFM12 mediated efficacious target cell lysis with a very fast on-set in vitro. Lysis induced by AFM11 was less efficacious (lower specific lysis than AFM12) but reproducibly more potent (lower EC50 value). In addition to the potency and efficacy of AFM11 and AFM12, different aspects of safety, such as effector cell activation in the presence and absence of target cells were investigated and will be described. Conclusions: Affimed's CD19/CD3 and CD19/CD16A TandAbs with identical anti-CD19 tumor-targeting domains but different effector cell-recruiting domains represent interesting molecules to study T-cell- or NK-cell-based immunotherapeutic approaches. The comparison of AFM11 and AFM12 demonstrated that AFM12-mediated lysis was fast and efficacious, whereas AFM11 showed a higher potency. In summary, the NK-cell recruiting TandAb AFM12 represents an alternative to T-cell recruiting molecules, as it may offer a different side effect profile, comparable to that of AFM13, the first NK-cell TandAb clinically investigated. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional Characterization of Gamma Delta T Cells and Allogeneic Activated NK Cells As Effector Cells for Tafasitamab (MOR208)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3801-3801
Author(s):  
Jung Hyun Her ◽  
Dominik Pretscher ◽  
Sungyoo Cho ◽  
Yu-Kyeong Hwang ◽  
Timm Hoeres ◽  
...  
Keyword(s):  
T Cells ◽  
Combination Therapy ◽  
B Cell ◽  
Nk Cells ◽  
Effector Cell ◽  
Γδ T Cells ◽  
Target Cells ◽  
Effector Cells

Introduction Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal antibody that binds to the human B cell surface antigen CD19. CD19 is broadly and homogeneously expressed across different B cell malignancies, including diffuse large B cell lymphoma (DLBCL), and amplifies B cell receptor signaling and induces tumor cell proliferation. Tafasitamab is currently in clinical development in patients with relapsed or refractory DLBCL in combination with the immunomodulatory drug lenalidomide (L-MIND study) and the chemotherapeutic agent bendamustine (B-MIND study). The modes of action of tafasitamab comprise antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity (apoptosis). Tafasitamab carries two amino acid exchanges in the Fc region to increase its affinity to Fcγ receptors, including FcγRIIIa. FcγRIIIa plays a key role in mediating ADCC and is expressed on the surface of natural killer (NK) cells, as well as the majority of γδ T cells. MG4101 (a novel therapeutic agent consisting of cryopreserved, ex vivo-expanded, highly activated NK cells) has demonstrated potent anticancer activity in preclinical in vitro and in vivo studies. Currently, MG4101 is in clinical development in patients with malignant lymphoma and advanced solid tumors. Here, we have characterized two FcγRIIIa receptor-expressing cell types, γδ T cells and NK cells (MG4101), as effector cells for tafasitamab in vitro and explored the concept of supplementing MG4101 during tafasitamab therapy using disseminated in vivo models of non-Hodgkin's lymphoma. Methods γδ T cells (CD3+/γδ T cell receptor+) were derived from different donors by stimulation of peripheral blood mononuclear cells with zoledronate/IL-2 for 9-10 days. These were applied as effector cells in in vitro ADCC assays with tafasitamab in Mino and Jeko-1 mantle cell lymphoma (MCL) cell lines, as well as primary patient-derived chronic lymphocytic lymphoma (CLL) and MCL cells. Further, effector cell activity of MG4101 was assessed using tafasitamab-mediated ADCC assays in Raji and Ramos Burkitt's lymphoma cells. The concept of combining tafasitamab with allogeneic effector cell therapy in vivo was studied in two therapeutic survival models of disseminated lymphoma in SCID mice. In the Raji model, tafasitamab (0.05 µg/mouse) was given on Day 1 after intravenous (IV) tumor inoculation, while MG4101 (2x107 cells/mouse) was given on Days 1, 3, 6, 8 and 10. In the Ramos model, tafasitamab (10 mg/kg) and MG4101 (2x107 cells/mouse) were applied twice weekly for 3 weeks starting on Days 3 and 4, respectively, after IV tumor inoculation. Results Tafasitamab in combination with γδ T cells showed distinctly increased ADCC in Mino and Jeko-1 target cells in vitro, compared with a negative control IgG1 antibody. ADCC assays with patient-derived CLL and MCL target cells confirmed tafasitamab-mediated cytotoxic activity and demonstrated a clear enhancement in activity compared with the non-Fc-enhanced version of tafasitamab that was unable to induce substantial cytotoxicity. In vitro ADCC assays with tafasitamab and MG4101 on Raji and Ramos cell lines confirmed potent effector cell activity of the ex vivo-expanded, cryopreserved, allogeneic NK cells. In the disseminated Raji survival model, combination therapy with a single low dose of tafasitamab (0.05 µg) and MG4101 resulted in a distinct increase in survival of the mice with an increased life span (ILS) of 100% compared with monotherapy (ILS of 57% for tafasitamab and 50% for MG4101). Of note, the combination demonstrated a substantial and more than additive enhancement in survival in the more therapeutic Ramos survival model (Figure 1). Combination therapy with tafasitamab (10 mg/kg) and MG4101 NK cells resulted in superior antitumor activity (ILS of 103%) compared with either tafasitamab monotherapy (ILS of 49%) or MG4101 alone (ILS of 25%). Conclusions FcγRIIIa-expressing immune cell types, including NK cells and γδ T cells, are potent effector cells for tafasitamab-mediated ADCC. Combination therapy with tafasitamab and allogeneic MG4101 NK cells in vivo demonstrated a more than additive survival benefit compared with tafasitamab or MG4101 monotherapy in a disseminated therapeutic lymphoma model. Combination of tafasitamab supplemented with immune effector cells could represent a promising new approach for lymphoma therapy. Disclosures Her: GC LabCell: Employment, Patents & Royalties. Cho:GC LabCell: Employment, Patents & Royalties. Hwang:GC LabCell: Employment, Equity Ownership, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties. Steidl:MorphoSys AG: Employment. Patra:MorphoSys AG: Employment. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties.

scholarly journals The in vitro effects of Bordetella pertussis lymphocytosis-promoting factor on murine lymphocytes. IV. Generation, characterization, and specificity of cytotoxic lymphocytes.

1979 ◽  
Vol 149 (6) ◽  
pp. 1393-1406 ◽  
Author(s):  
A S Kong ◽  
S I Morse
Keyword(s):  
Bordetella Pertussis ◽  
Tumor Rejection ◽  
Cytotoxic Lymphocytes ◽  
Normal Cells ◽  
Effector Cells ◽  
Lymph Node Cells ◽  
In Vitro Effects ◽  
Cytotoxic Effector

Cytotoxic effector lymphocytes were induced in cultures of mouse spleen or lymph node cells by lymphocytosis promoting factor (LPF). The LPF-activated cytotoxic cells: (a) were not generated unless proliferation occurred; (b) sedimented in the lighter density fraction of a bovine serum albumin gradient; (c) were large, blast-like cells; and (d) were lysed by Thy-1.2 antiserum plus complement and, therefore, were T cells. Neither LPF alone nor supernates from stimulated cultures were cytotoxic. Unlike the situation with concanavalin A and phytohemagglutinin P, LPF-stimulated cytotoxic effector lymphocytes required no further addition of mitogen for maximal cytotoxicity. The effector cells displayed specificity, destroying only allogeneic but not syngeneic normal cells; in the case of tumor cells, both allogeneic and syngeneic cells werelysed in the absence of added mitogen. The reason for differentiated cytotoxicity toward syngeneic tumor and normal cells is not clear but may have some relevance to in vivo tumor rejection initiated by Bordetella pertussis.

scholarly journals Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response.

1994 ◽  
Vol 180 (1) ◽  
pp. 383-387 ◽  
Author(s):  
J Lesley ◽  
N Howes ◽  
A Perschl ◽  
R Hyman
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Surface Receptor ◽  
Target Cells ◽  
Cd44 Isoforms ◽  
Effector Cells ◽  
Binding Function ◽  
Cytotoxic Effector

Though CD44 functions as a cell surface receptor for hyaluronan (HA) in some cell lines, most normal hematopoietic cells expressing CD44 do not bind HA. Certain CD44-specific monoclonal antibodies (mAbs) can rapidly induce CD44-mediated HA binding in normal murine T cells. This observation suggests that in vivo mechanisms may exist for activating the HA receptor function of CD44 on normal T cells. Here, it is shown that up to one third of splenic T cells are capable of CD44-mediated binding of fluorescein-conjugated HA (Fl-HA) during an in vivo allogeneic response. HA binding activity peaks at 7-8 d postinjection and declines rapidly. These rapid kinetics could be the result of transient activation of CD44 function and/or differentiation or expansion of short-lived population(s) that have constitutive HA-binding function. Both CD4 and CD8 T cells are included in the HA binding population which is strongly CD44 positive. After separation of HA-binding cells from nonbinding cells by cell sorting, it is shown that almost all cytotoxic effector cells are found in the HA-binding population. However, there is no evidence that CD44-mediated HA recognition is directly involved in the killing of target cells, since cytotoxicity could not be inhibited by CD44-specific mAbs that inhibit HA binding or by soluble HA. PCR amplification of cDNA reverse transcribed from RNA of sorted HA-binding cells indicated no evidence for CD44 isoforms other than the standard (hematopoietic) form. Though CD44 expression is known to be elevated upon T cell activation, and, as shown here, HA-binding function is induced in a portion of CD44-expressing T cells including cytotoxic effector cells, the role of CD44 and HA-recognition in immune responses is not known.

scholarly journals Cell-mediated lympholysis to H-2-matched target cells modified with a series of nitrophenyl compounds.

1976 ◽  
Vol 144 (4) ◽  
pp. 1134-1140 ◽  
Author(s):  
T G Rehn ◽  
J K Inman ◽  
G M Shearer
Keyword(s):  
T Cell ◽  
Target Cells ◽  
Cell Recognition ◽  
Receptor Model ◽  
Spleen Cells ◽  
Effector Cells ◽  
T Cell Recognition ◽  
Cytotoxic Effector

The specificity of C57BL/10 cytotoxic effector cells generated by in vitro sensitization with autologous spleen cells modified with a series of related nitrophenyl compounds was investigated. The failure of trinitrophenyl (TNP)-sensitized effector cells to lyse TNP-beta-alanylglycylglycyl(AGG)-modified target cells is presented as evidence contradicting the intimacy or dual receptor model or T-cell recognition in its simplest form. Data are also shown indicating that sensitization with N-(3-nitro-4-hydroxy-5-iodophenylacetyl)-AGG-modified stimulating cells generates noncross-reacting clones of cytotoxic effector cells.

scholarly journals In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Béatrice Clémenceau ◽  
Sandrine Valsesia-Wittmann ◽  
Anne-Catherine Jallas ◽  
Régine Vivien ◽  
Raphaël Rousseau ◽  
...  
Keyword(s):  
Chimeric Antigen Receptor ◽  
Tumor Regression ◽  
Critical Role ◽  
Antigen Receptor ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Her2 Positive ◽  
Positive Target

The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ(NK-92CD16) or a trastuzumab-based scFv-FcεRIγchimeric receptor (NK-92CAR). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition byNK-92CD16was always inferior to that observed after direct recognition byNK-92CAR. In contrast, and somehow unexpectedly, in vivo, adoptive transfer ofNK-92CD16+ trastuzumab but not ofNK-92CARinduced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of theNK-92CARin the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

scholarly journals Participation of the H-2 antigens of tumor cells in their lysis by syngeneic T cells.

1976 ◽  
Vol 143 (3) ◽  
pp. 601-614 ◽  
Author(s):  
J W Schrader ◽  
G M Edelman
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Target Cell ◽  
Cytotoxic T Cells ◽  
Hybrid Origin ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Effector Cells

Cytotoxic T lymphocytes were generated in vitro against H-2 compatible or syngeneic tumor cells. In vitro cytotoxic activity was inhibited by specific anti-H2 sera, suggesting that H-2 antigens are involved in cell lysis. Two observations directly demonstrated the participation of the H-2 antigens on the tumor cells in their lysis by H-2-compatible T cells. First, coating of the H-2 antigens on the target tumor cell reduced the number of cells lysed on subsequent exposure to cytotoxic T cells. Second, when cytotoxic T cells were activated against an H-2 compatible tumor and assayed against an H-2-incompatible tumor, anti-H-2 serum that could bind to the target cell, but not to the cytotoxic lymphocyte, inhibited lysis. H-2 antigens were also shown to be present on the cytotoxic lymphocytes. Specific antisera reacting with these H-2 antigens, but not those of the target cell, failed to inhibit lysis when small numbers of effector cells were assayed against H-2-incompatible target cells or when effector cells of F1-hybrid origin and bearing two H-2 haplotypes were assayed against a tumor cell of one of the parental strains. These findings suggest that it is the H-2 antigens on the tumor cell and not those on the cytotoxic lymphocytes that are important in cell-mediated lysis of H-2-compatible tumor cells.

scholarly journals T cells specific for hapten-modified self are precommitted for self major histocompatibility complex antigens before encounter with the hapten.

1978 ◽  
Vol 147 (4) ◽  
pp. 1065-1077 ◽  
Author(s):  
C A Janeway ◽  
P D Murphy ◽  
J Kemp ◽  
H Wigzell
Keyword(s):  
T Cells ◽  
Suppressor Cells ◽  
The Other ◽  
Target Cells ◽  
Killer Cells ◽  
Effector Cells ◽  
Complex Cells ◽  
Histocompatibility Complex ◽  
Peripheral T Cells ◽  
Cytotoxic Effector

The technique of antigen-driven, 5-bromo-deoxyuridine and light suicide has been adapted to eliminate the precursors of cytotoxic effector cells both for alloantigen and for 2,4,6-trinitrophenyl(TNP)-modified stimulator and target cells. Using this technique, the following observations have been made. Precursors of killer cells specific for alloantigen can be suicided independently of precursors of killer cells specific for TNP-modified self cells. The loss of activity during this procedure is not due to either specific or nonspecific suppressor cells, as judged by mixing experiments. With responder cells from F1 animals, it has been possible to show that precursors specific for TNP-modified cells from one parent are suicided independently of precursors specific for TNP-modified cells of the other parent, but only if the parental strains differ in the K and D regions of the H-2 complex. Cells of F1 mice derived from K and D identical, I region different, parental strains were specifically suicided by TNP-modified stimulator cells from either parent. However, the cross-reactive killing of TNP-self targets induced by stimulation with allogeneic cells is not eliminated by first suiciding with TNP-parental cells, suggesting that the precursors of these two types of TNP-self killer cells are different. This is compatible with reported differences in their specificity, as confirmed in this report. Finally, deletion of alloreactive cells by this technique reveals little or no reactivity specific for TNP-modified allogeneic stimulator cells. In summary, these results strongly suggest that recognition of self MHC antigens is preprogrammed in peripheral T cells of normal animals, and is not acquired during the immunization process. They also suggest that cells specific for modified alloantigen are relatively rare in the strains of mice studied.

A Non-Internalizing Anti-CD40 Antibody, CHIR-12.12, Blocks CD40L-Induced Cytokine Production and Mediates Greater ADCC Than Rituximab in Primary CLL Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2964-2964
Author(s):  
Xia Tong ◽  
Sharon Lea Aukerman ◽  
Karen Lin ◽  
Natasha Aziz ◽  
Cheryl Goldbeck ◽  
...  
Keyword(s):  
Cell Surface ◽  
B Cell ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
Adcc Activity ◽  
Effector Cells ◽  
Cells Cultured

Abstract CD40 is expressed on chronic lymphocytic leukemia (CLL) cells, and CD40 activation leads to signaling critical for cell survival and proliferation. We have previously described a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, generated in XenoMouse® mice (Abgenix, Inc.), and have demonstrated that it inhibits normal human B cell proliferation and survival and mediates potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. In this study, we examined the ability of CHIR-12.12 to modulate cytokine production by primary CLL cells and compared the ADCC activity of CHIR-12.12 with rituximab against primary CLL cells. Primary CLL cells stimulated with CD40L produced a variety of cytokines, including IL-10, TNF-α , IL-8, GM-CSF, IL-6, MCP-1, and MIP-1β. Addition of CHIR-12.12 to primary CLL cells inhibited CD40L-mediated production of these cytokines. Cytokine production by primary CLL cells cultured with CHIR-12.12 alone in the absence of CD40L did not exceed levels produced by CLL cells cultured in medium. These data suggest that CHIR-12.12 is a potent antagonist for CD40L-mediated cytokine production by primary CLL cells and shows no agonistic activity by itself. We next compared the relative ADCC activity of CHIR-12.12 and rituximab against ex vivo primary CLL cells from 8 patients. CHIR-12.12 exhibited greater ADCC than rituximab against CLL cells from all patients. The average percent of maximum lysis by CHIR-12.12 and rituximab were 49 ± 16% and 31 ± 14%, respectively. CHIR-12.12 was greater than 10-fold more potent than rituximab, as measured by ED50 values (14.1 pM versus 155.5 pM, respectively). Quantitative CD20 and CD40 density on CLL cells and the degree of antibody internalization were investigated as potential reasons for the difference in ADCC activity. The greater ADCC potency and efficacy of CHIR-12.12 was not dependent on a higher density of cell surface CD40 molecules, as there were 1.3 to 14-fold higher numbers of CD20 than CD40 molecules on the cell surface. Antibody internalization studies using primary CLL cells conducted by flow cytometry and confocal microscopy show that upon binding to CD40 at 37°C, CHIR-12.12 remains uniformly distributed on the cell surface, even after 3 hours. In contrast, after binding at 37°C, rituximab is redistributed into caps and internalized. These data suggest that the potent ADCC activity of CHIR-12.12 may be partly related to its ability to remain on the surface of target cells uniformly, allowing optimal interaction with effector cells. Taken together, these results suggest that CHIR-12.12 may be effective at mediating potent ADCC against CLL cells in vivo. CHIR-12.12 is currently in Phase I trials for B-cell malignancies.

Loss of CD20 Expression and Exhaustion of Effector Cells Limit ADCC in CLL Patients Treated with Rituximab.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1610-1610 ◽  
Author(s):  
Berengere Vire ◽  
Justin SA Perry ◽  
Elinor Lee ◽  
Lawrence S Stennett ◽  
Leigh Samsel ◽  
...  
Keyword(s):  
Nk Cells ◽  
Effector Cell ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Effector Cells ◽  
Cell Functions ◽  
Anti Cd20

Abstract Abstract 1610 Poster Board I-636 A major mechanism how the chimeric anti-CD20 monoclonal antibody rituximab (RTX) depletes B-cells is antibody-dependent cellular cytotoxicity (ADCC). ADCC has been modeled in-vitro and in mouse models. However, investigations on ADCC directly in patients treated with RTX are scarce. Recent efforts have focused on improving ADCC through modifications in the Fc binding portion of novel antibodies or through stimulation of effector cell functions with GM-CSF. A more detailed understanding of ADCC as a therapeutic process is needed to optimize such strategies and to identify biomarkers of improved efficacy. Here we report a comprehensive analysis of ADCC in previously untreated CLL patients during the first two RTX infusions (375mg/m2) given in combination with fludarabine every 4 weeks. Following the initial infusion of RTX the absolute lymphocyte count (ALC) decreased by a median of 74% at 2h, followed by a partial recrudescence of cells so that by 24h the median decrease in ALC reached 39% (n=11). ADCC is mediated by effector cells that include NK cells, monocytes/macrophages, and granulocytes. First, we investigated changes in NK cell function: consistent with NK cell activation we found an increase in CD69 at 2, 6 and up to 24h (median 4.2-fold, p=0.005, n=10) after RTX administration and increased expression of the degranulation marker CD107a/b (median 1.9-fold, p<0.001, n=5) and down-regulation of perforin expression (median decrease 63%, p<0.001, n=5) at 4h from treatment start. Activation of NK cells is triggered by the engagement of CD16/FcγRIIIa by RTX coated CLL cells. Interestingly, CD16 expression on NK cells was rapidly lost, already apparent at 2h and maximal at 6h from the start of the RTX infusion (median decrease 82%, p=0.02, n=10) and was not completely recovered by 24h. We also found a significant decrease in expression of CD16 on granulocytes (78%, p<0.001, n=5) but an increase in monocytes (3.9-fold, p<0.001, n=5). In addition to loss of CD16, we found that the cytotoxic capacity of the effector cells was rapidly exhausted: in an oxidative-burst assay, monocytes showed a significant decrease in the production of reactive oxygen species 4h after initiation of RTX infusion (median 60% decrease, p=0.043) and at 6h from the start of the RTX infusion NK cell-mediated killing of K562 target cells was reduced by half (p<0.001, n=3). Interestingly, both the acute reaction to RTX infusions that manifest as a cytokine release syndrome and changes in effector cell function peaked during the first hours of the RTX infusion. We hypothesized that this might be due to the process of CD20 shaving, a rapid and pronounced decrease of CD20 cell surface expression modeled in-vitro and in mice as the result of a mechanism called trogocytosis that relies on the direct and rapid exchange of cell membrane fragments and associated molecules between effectors and target cells (Beum, J Immunol, 2008). First, we used western blot analysis of total CD20 protein in CLL cells and found a rapid loss of CD20 that was apparent already at 2h resulting in virtually complete loss of expression at 24h. Next, we used ImageStream technology to directly visualize ADCC interactions in-vivo. We indeed detected transfer of CD20 from CLL cells to NK cells and monocytes, resulting in complete CD20 loss in circulating CLL cells. While we detected transfer of CD20 into both cell types, monocytes were much more engaged in trogocytosis than NK cells. Consistently, 4h post RTX infusion we found a significant increase in intracellular RTX in granulocytes and monocytes using intracellular staining for human IgG. CD20 shaving appears to be of particular importance given that immunohistochemical analyses revealed that persistent disease in the bone marrow aspirates after 4 cycles of RTX treatment was mostly CD20 negative. Collectively, our results identify loss of CD20 from CLL cells by trogocytosis and exhaustion of immune effector mechanisms as limitations for anti-CD20 immunotherapy. These data identify possible avenues for improving CD20 mediated immunotherapy and characterize endpoints on which different anti-CD20 antibodies can be compared. Given that trogocytosis appears to be a common occurrence our findings likely have general importance to immunotherapy of hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

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